1. Field of the Invention
The invention relates to reagents and methods for enhancing efficacy of prodrugs. Specifically, the invention relates to targeted delivery of enzymes, in particular specifically engineered enzymes, to cells in need thereof, particularly for converting prodrugs to chemotherapeutically active drugs. The invention is particularly directed to targeted delivery of said enzymes to cancer cells of specific tumor types. Specifically, the invention provides genetically-engineered modified human deoxycytidine kinase (dCK) with enhanced activity towards nucleoside analogs used in cancer chemotherapy.
2. Background of the Related Art
Many currently-used chemotherapeutic agents are members of a class of drugs referred to as anti-metabolites. One type of such anti-metabolite are molecules that block or subvert one or more of the metabolic pathways involved in DNA synthesis by mimicking naturally-occurring nucleic acid building blocks. Many of this type of anti-metabolite are nucleoside analogs (NAs). These NAs themselves usually do not possess any therapeutic activity (and thus are properly termed prodrugs) and rely on their conversion, for the most part to the triphosphorylated form, to become active (prodrug-to-drug metabolism).
The efficiency of conversion from the administered nucleoside to the active triphosphorylated form determines the efficacy of these types of prodrugs. The serial phosphorylation of NAs to their triphosphorylated metabolite, via monophosphate and diphosphate intermediates, is catalyzed by human cellular kinases, with deoxycytidine kinase (dCK) playing a major role. dCK transfers a phosphoryl group from ATP (or other triphosphorylated nucleotides) to deoxycytidine (dC). Native dCK has been shown to localize in the cytoplasm, though over-expression of dCK from a transfected construct may result in nuclear localization of dCK (Hatzis et al., 1998, Journal Biol. Chem. 273:30239-30243). dCK is required for the phosphorylation of numerous NAs used in chemotherapy, including AraC (1-β-D-arabinofuranosylcytosine; Cytarabine), dFdC (2′,2′-difluorodeoxycytidine; Gemcitabine), FaraA (2-fluoro-9-β-D-arabinosyladenine; Fludarabine) and 2CdA (2-chlorodeoxyadenosine, Cladribine) (Van Rompay et al., 2000, Pharmacol. Ther. 87:189-98). Therefore, the activity of dCK is one of the factors that determine the sensitivity of cancer (including leukemias and certain solid tumors) to deoxynucleoside toxicity and hence, therapy (Stegmann et al., 1995, Blood 85:1188-94; Lotfi et al., 1999, Clin. Cancer Res. 5:2438-44; Kakihara et al., 1998, Leuk. Lymphoma 31:405-9; Bergman et al., 1999, Biochem. Pharmacol. 57:397-406; Goan et al, 1999, Cancer Res. 59:4204-7).
There is a direct correlation between dCK enzymatic activity in tumor cell lines and the sensitivity of those cells to the toxicity of nucleoside analog chemotherapeutic prodrugs (Hapke et al., 1996, Cancer Res. 56:2343-7). Cells lacking dCK activity are resistant to a variety of drugs, including ara-C, cladribine, fludarabine and gemcitabine (Owens et al., 1992, Cancer Res. 52:2389-93; Ruiz van Haperen et al., 1994, Cancer Res. 54:4138-43) and drug sensitivity to ara-C can be restored by expressing functional dCK protein in cells that do not express this enzyme natively or in which only mutationally-inactivated forms thereof are expressed (Stegmann et al., 1995, Blood 85:1188-94). Moreover, in vivo studies conducted on animal tumors using gemcitabine showed that increased dCK activity, mediated by dCK gene transfer, resulted in enhanced accumulation and prolonged elimination kinetics of gemcitabine triphosphate, and ultimately, to a better tumor response to the prodrug (Blackstock et al., 2001, Clin. Cancer Res. 7:3263-8).
More efficient prodrug-to-chemotherapeutic drug conversion, i.e. from NA to NA-triphosphate, would greatly increase the potency of such prodrugs and reduce undesired side effects common in chemotherapeutic treatments (due at least in part to higher concentrations of the drug needed to achieve a therapeutic result, with concomitant toxicity to normal cells and tissues). Higher concentrations of the active metabolites of nucleoside analog prodrugs, particularly in the cancer cells themselves, would result in a better therapeutic index for these prodrugs. In addition, some tumor cells develop resistance to chemotherapeutic agents that are administered as prodrugs and activated by enzymes expressed in target tumor cells, by reducing or eliminating expression of the gene(s) encoding the enzymatic activity. Resistance arising from such down-regulation of cellular gene expression could be overcome by targeted delivery of the enzyme directly to the tumor cell. Additionally, targeted delivery into cancer cells of a modified enzyme that has acquired additional substrate specificity as compared to the wild type enzyme allows the use of additional NAs to treat the targeted cancer cells. Thus, there is a need for more efficient enzymes and enzymes that utilize a wider variety of substrate NAs, especially NAs that are not normally phosphorylated by wild-type cellular enzymes, and for methods of using these enzymes to treat cancer cells.